Pharmaceutical composition for enhancing immunity, and extract of poria

ABSTRACT

A pharmaceutical composition is used to enhance immunity of the human body. The composition contains potent components of lanostane compounds. A method is devised to obtain an extract from metabolite, sclerotium, or fermentation product of  Poria cocos  (Schw) Wolf. The extract contains 5-60% of the lanostane compounds by weight of the extract. The extract is devoid of secolanostane capable of inhibiting immunity development.

RELATED APPLICATIONS

This is a divisional application of U.S. application Ser. No. 10/717,559, filed Nov. 21, 2003; which claims priority to Taiwan, Republic of China Application No. 92113393, filed May 16, 2003. All of the above applications are incorporated into the current divisional application by reference.

FIELD OF THE INVENTION

The present invention relates to a pharmaceutical composition containing lanostane as a potent component thereof for enhancing immunity. The present invention further relates to an extract of Poria cocos (Schw) Wolf for the purpose of immunity enhancement.

BACKGROUND OF THE INVENTION

The Poria extract has tonic effect, as well as a smoothing effect on stomach disorder. According to the Chinese medicine, the Poria extract is classified as a tranquilizer and a uretic agent. In addition, the Poria extract is used as one of essential ingredients of the Chinese medicine prescription for vital activity. On the basis of researches and experiments, which have been conducted in recent years on the pharmacological effect of the Poria extract, it has been concluded that the Poria extract has a favorable effect on tumor prevention, and that the Poria extract is beneficial to immunity enhancement and gastrointestinal system of a person suffering from a chronic disease.

As exemplification, the Japan Patent Publication Numbers 55-111791 and 57-38794 disclose an extract which is obtained from the cultivated mycelia of Poria cocos (Schw) Wolf and is effective in tumor prevention. The Japan Patent Publication No. 55-111422 discloses an extract which is directly obtained from Poria cocos (Schw) Wolf for use in tumor prevention. The Japan Patent Publication No. 8-119864 discloses an extract which is obtained by extracting Poria cocos (Schw) Wolf with methanol. By separation, triterpene compounds such as lanostanes and secolanostanes are obtained from the extract and are used as anti-emetic agents. The Japan Patent Publication No. 9-025232 discloses triterpene compounds, which is obtained by extracting Poria cocos (Schw) Wolf with methanol. The compounds are useful as a tumor promotion-inhibiting agent. The Japan Patent Publication No. 9-176184 discloses an extract of Poria cocos (Schw) Wolf, which is then refined to produce triterpene compounds for use as an agent for inhibiting inflammation and tumor promotion.

The China Patent Publication No. 1008183 discloses a method of making a Poria extract containing triterpene compounds. The method involves extracting the Poria powder with an acidic alcohol, neutralizing the extract with a basic solution, concentrating the neutralized solution, adjusting the pH thereof to about 10 and filtering the solution, acidifying the filtrate to form a precipitate, washing the precipitate after filtration, and drying the washed precipitate. The Poria extract so obtained is found to have a tumor-preventive effect and an immunity activation effect.

SUMMARY OF THE INVENTION

The primary objective of the present invention is to provide a method of prepare a potent component having an improved biological activity from Poria cocos (Schw) Wolf, and a Poria extract containing the potent component.

It is another objective of the present invention to provide a method of using Poria cocos (Schw) Wolf to prepare a potent component capable of enhancing immunity of mammal, and a Poria extract containing the Potent component.

It is still another objective of the present invention to provide a new use of lanostane. One aspect of this use is a pharmaceutical composition for enhancing immunity of mammal. A pharmaceutical composition embodied according to the present invention contains one or more lanostane compounds. The pharmaceutical composition is used to adjust or enhance immunity and can be dispensed by a dermal, oral, or hypodermic administration, and may be in a slow-release dosage form.

The present invention makes use of the immunity experiments to verify the pharmacological properties of the potent component of the Poria extract. The potent component is a low polarity portion (PCM), which contains major compounds of K1, K2, K3, K4, and trace of K4a, K4b, K5, K6a, K6b, which are all lanostane compounds. The compounds of K2, K3, and K4 have an immunity enhancement effect.

The method of the present invention makes use of the conventional extraction process, by means of which a crude extract is obtained. A chromatographic separation is used to separate constituents of the crude extract, which include a lanostane fraction and a secolanostane fraction. The lanostane fraction is relatively smaller in polarity than the secolanostane fraction. The lanostane fraction is obtained by using an eluent made of dichloromethane:methanol=96:4, whereas the secolanostane fraction is obtained by using an eluent made of dichloromethane:methanol=90:10 or 0:100. The position of the lanostane fraction is identified by the thin layer chromatography, which has a chromatographic value (Rf) being≧0.1 when a developing solution of dichloromethane:methanol (96:4) is used. The chromatographic value of the secolanostane fraction is smaller than 0.1. By a silica gel column chromatography, the lanostane fraction is separated into several lanostane compounds with an eluent made of dichloromethane:methanol alcohol=97:3 to 95:5.

According to the method of the present invention, 2.6 grams of PCM are obtained from one kilogram of Poria cocos (Schw) Wolf. According to the method of the China Patent Publication No. 1008183, three grams of a crude extract are obtained from one kilogram of Poria cocos (Schw) Wolf. When this crude extract is purified by the method of the present invention, only one gram of PCM is obtained.

The features of the present invention will be more readily understood upon a thoughtful deliberation of the following detailed description of the nonrestrictive embodiments of the present invention with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flow diagram of a method of the present invention for making lanostane compounds from Poria cocos (Schw) Wolf.

FIG. 2 shows a flow diagram of making a Poria extract in accordance with a method disclosed by the China Patent Publication No. 1008183, and a low polarity portion of the Poria extract according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

These inventors of the present invention repeated the example disclosed in the China Patent Publication No. 1008183 by using Poria cocos (Schw) Wolf, which was grown in China. By an acid extraction and alkaline/acidic treatments, 3 grams of a crude Poria extract were obtained from one kilogram of Poria cocos (Schw) Wolf. This is in agreement with the range of 2.5 g±0.5 g disclosed in the afore-mentioned China Patent Publication. As a result of further separation, 400 mg of purified lanostane compounds were obtained. In another words, the extract obtained according to the method disclosed in the afore-mentioned China Patent publication contains about 13% of lanostane fraction, with 87% of the extract being the secolanostane fraction and other unidentified constituents.

As a result of further experiments carried out by these inventors of the present invention, the lanostane fraction has no toxic effect on vat spleen cells and is pharmacologically effective. The secolanostane fraction has a toxic effect on rat spleen cells.

The present invention discloses a pharmaceutical composition capable of enhancing immunity of mammal, such as Homo sapiens. The composition contains a therapeutically effective amount of lanostane having the following chemical formula (I) as an active ingredient, and a pharmaceutically acceptable carrier or diluent for the active ingredient:

wherein R₁ is either H or CH₃; R₂ is OCOCH₃, ═O or OH; R₃ is H or OH; R₄ is —C(═CH₂)—C(CH₃)₂R_(a), wherein R_(a) is H or OH, or —CH═C(CH₃)—R_(b), wherein R_(b) is CH₃ or CH₂OH; R₅ is H or OH; and R₆ is CH₃ or CH₂OH.

Preferably, the pharmaceutical composition comprises 0.1-60% of the lanostane (I) by weight of the composition and is orally administered.

The present invention also discloses a Poria extract capable of enhancing immunity of mammal. The extract contains 5-60%, preferably 10-20%, of the lanostane (I) by weight of the extract. The extract is substantially devoid of secolanostane.

The present invention covers a method for the preparation of the Poria extract. The method includes a first step in which the metabolites, the fermentation products, and the sclerotium of Poria cocos (Schw) Wolf are extract by a solvent, such as water, methanol, ethanol, or a mixture thereof, thereby resulting in production of a liquid extract, which is then concentrated to form a concentrated substance. The concentrated substance is introduced into a silica gel column, and is eluted with an eluent having a low polarity. As a result, an eluate is produced and collected. The eluate is concentrated to form a concentrated eluate, which has a chromatographic value (Rf)≧0.1 in accordance with a thin layer chromatography, which is developed by a mixed solvent of dichloromethane:methanol=96:4 and is detected by an ultraviolet lamp and iodine vapor.

It is suggested that the extraction is carried out by using 95% ethanol.

Preferably, the concentrated substance is further extracted with a two-phase solvent containing methanol and n-hexane in a volumetric ratio of 1:1. The methanol layer is separated and is concentrated to form a concentrate, which is used as a feed to the silica gel column.

It is recommended that the low polarity eluent is a mixed solvent containing dichloromethane and methanol in a volumetric ratio of 96.5:3.5.

Preferably, the lanostane (I) has the following structures:

The present invention also discloses a method of enhancing immunity of an individual comprising administering to the individual a therapeutically effective amount of the lanostane (I) of the present invention.

The present invention also discloses a method of enhancing immunity of an individual comprising administering to the individual a therapeutically effective amount of the Poria extract of the present invention.

Example 1

As illustrated in FIG. 1, a Poria powder was made of 30 kilograms of the China-grown Poria cocos (Schw) Wolf. The Poria powder was extracted with 120 L 95% alcohol for 24 hours. The mixture was filtered to obtain a filtrate. The residue was extracted and filtered for another three cycles. The filtrates were combined and concentrated to bring about a dried extract in amount of 265.2 grams. The dry extract was undergone a distribution extraction with a two-phase extraction agent (n-hexane:95% methanol=1:1), and the methanol layer was removed therefrom, which is then concentrated to obtain a dry solid in an amount of 246.9 grams. A separation of the dry solid was carried out by means of a silica gel column, which was filled with silica gel 10-40 times of the weight of the dry solid. The silica gel having a diameter of 70-230 mesh was made by Merck corporation with a code of Silica Gel 60. The column was eluted by the following eluates in sequence: a mixed solvent of dichloromethane:methanol=96:4; a mixed solvent of dichloromethane:methanol=90:10, and pure methanol. The eluates were tested by the thin layer chromatography (TLC), wherein an ultraviolet lamp and iodine vapor were used for detecting, and a mixed solvent of dichloromethane:methane=96:4 was used as a developing liquid. The eluates having similar constituents in the TLC were combined.

The elution carried out with the mixed solvent of dichloromethane:methanol=96:4 resulted in a PCM portion in amount of 78 grams. The PCM shows 6 trace points in the thin layer chromatography. The resulting eluates from the elutions carried out with the eluents of dichloromethane:methanol=90:10 and pure methanol were combined to obtain a PCW portion in amount of 168 grams.

The PCM portion was further separated by means of an eluent of dichloromethane:methanol=96.5:3.5 and the silica gel column. With the thin layer chromatography, the eluates were collected as three fractions, which were respectively K1 (Rf=0.64), PCM-1 (containing trace K1, K3 (Rf=0.55), K5 (Rf=0.49)), and PCM-2 (containing K2 (Rf=0.30), K4 (Rf=0.24), K6 (Rf=0.19). The K1 fraction (3.5 g) was further subjected to a high performance liquid chromatography (HPLC) using a carbon-18 column and a mobile phase of methanol-water (90:10)). 3.0 grams of K1 component was obtained.

With the same HPLC and a mobile phase of methanol-water (87:13), the PCM-1 fraction was separated into K3, K5, and trace K1 components. The K3 component was further purified to yield K3 (1.93 g) by means of the same HPLC and a mobile phase of methanol-water (84:16). The K5 component was further purified with the same HPLC and two mobile phases of methanol-water (93:7) and (91:9) in sequence to yield K5 (47.6 mg).

With the same HPLC and a mobile phase of methanol-water (87:13), the PCM-2 fraction were separated into K6 trace component (K6a+K6b), K4 trace component (K4a+K4b), K2 component, and K4 component. By making use of the same HPLC and a mobile phase of methanol-water (84:16), the K2 component and the K4 component were further purified to yield K2 (6.2 g) and K4 (0.55 g).

With the same HPLC and a mobile phase of CH₃CN-water (68:32), the K6 trace component was purified to yield K6a (21.4 mg) and K6b (90.7 mg). The K4 trace component was subjected to the same HPLC and a mobile phase of methanol-water (76:24) to yield K4a (66.0 mg) and K4b (86.8 mg).

The afore-mentioned K1-K6 compounds have analytical data as follows:

K1: mixture, EI-MS: major component, 528[M]⁺; trace component, 526[M]⁺ K1 (major component): ¹³C-NMR (0:35.4 (c-1), 24.5 (c-2), 80.6 (c-3), 38.0 (c-4), 50.7 (c-5), 18.4 (c-6), 26.8 (c-7), 135.0 (c-8), 134.4 (c-9), 37.2 (c-10), 20.9 (c-11), 29.7 (c-12), 48.8 (c-13), 46.3 (c-14), 43.6 (c-15), 26.6 (c-16), 57.3 (c-17), 17.8 (c-18), 19.2 (c-19), 48.6 (c-20), 178.6 (c-21), 31.6 (c-22), 33.2 (c-23), 156.1 (c-24), 34.1 (c-25), 22.0 (c-26), 21.9 (c-27), 28.0 (c-28), 16.8 (c-29), 25.4 (c-30), 107.0 (c-31), 21.1 (CH₃COO—), 170.5 (CH COO—)

K1 (trace component): ¹³C-NMR (δ c): 35.6 (c-1), 24.5 (c-2), 80.6 (c-3), 37.8 (c-4), 49.7 (c-5), 23.1 (c-6), 120.6 (c-7), 142.8 (c-8), 145.8 (c-9), 37.6 (c-10), 117.0 (c-11), 36.3 (c-12), 49.4 (c-13), 45.1 (c-14), 44.4 (c-15), 76.4 (c-16), 57.6 (c-17), 17.6 (c-18), 22.8 (c-19), 48.4 (c-20), 178.5 (c-21), 31.4 (c-22), 33.2 (c-25), 156.0 (c-24), 34.1 (c-25), 22.0 (c-26), 21.9 (c-27), 28.2 (c-28), 17.1 (c-29), 26.5 (c-30), 107.0 (c-31), 21.1 (CH₃COO—), 170.4 (CH₃COO—)

K2: mixture, EI-MS: major component, 486[M]⁺; trace component, 484[M]⁺ K2 (major component): ¹³C-NMR (δ c): 36.6 (c-1), 29.1 (c-2), 78.5 (c-3), 40.0 (c-4), 51.4 (c-5), 19.2 (c-6), 27.4 (c-7), 135.4 (c-8), 135.3 (c-9), 37.9 (c-10), 21.4 (c-11), 30.2 (c-12), 49.3 (c-13), 46.7 (c-14), 44.2 (c-15), 77.1 (c-16), 57.8 (c-17), 18.2 (c-18), 19.8 (c-19), 49.2 (c-20), 179.4 (c-21), 32.1 (c-22), 33.7 (c-23), 156.5 (c-24), 34.6 (c-25), 22.5 (c-26), 22.4 (c-27), 29.1 (c-28), 16.8 (c-29), 25.9 (c-30), 107.5 (c-31)

K2 (trace component): ¹³C-NMR (δ c): 36.7 (c-1), 29.1 (c-2), 78.5 (c-3), 40.0 (c-4), 49.8 (c-5), 24.3 (c-6), 121.2 (c-7), 143.3 (c-8), 145.2 (c-9), 38.0 (c-10), 118.1 (c-11), 37.2 (c-12), 45.5 (c-13), 49.1 (c-14), 44.8 (c-15), 76.8 (c-16), 58.0 (c-17), 18.1 (c-18), 22.9 (c-19), 48.0 (c-20), 179.4 (c-21), 31.9 (c-22), 33.7 (c-23), 156.5 (c-24), 34.6 (c-25), 22.6 (c-26), 22.4 (c-27), 29.1 (c-28), 16.8 (c-29), 26.8 (c-30), 107.5 (c-31)

K3: mp: 278-280° C.

[α]_(D) ²⁴+3° (c 0.6, Pyridine)

EI-MS m/z: 482[M]⁺, ¹³C-NMR (δ c): 37.5 (c-1), 35.7 (c-2), 216.7 (c-3), 48.3 (c-4), 51.8 (c-5), 24.6 (c-6), 121.4 (c-7), 143.5 (c-8), 145.4 (c-9), 38.2 (c-10), 118.3 (c-11), 36.9 (c-12), 45.7 (c-13), 50.0 (c-14), 44.9 (c-15), 77.2 (c-16), 58.1 (c-17), 18.3 (c-18), 22.7 (c-19), 49.2 (c-20), 179.6 (c-21), 32.0 (c-22), 33.8 (c-23), 156.7 (c-24), 34.8 (c-25), 22.7 (c-26), 22.6 (c-27), 26.3 (c-28), 23.1 (c-29), 27.1 (c-30), 107.8 (c-31)

K4: mp:>300° C.

[α]_(D) ²⁴+18° (c 0.5, Pyridine)

EI-MS m/z: 484[M]⁺, ¹³C-NMR (δ c): 31.4 (c-1), 27.4 (c-2), 76.0 (c-3), 38.6 (c-4), 44.5 (c-5), 24.2 (c-6), 122.0 (c-7), 143.5 (c-8), 147.4 (c-9), 38.6 (c-10), 116.9 (c-11), 37.0 (c-12), 45.9 (c-13), 50.2 (c-14), 45.1 (c-15), 77.3 (c-16), 58.2 (c-17), 18.4 (c-18), 23.7 (c-19), 49.3 (c-20), 179.8 (c-21). 32.1 (c-22), 33.9 (c-23), 156.7 (c-24), 34.9 (c-25), 22.8 (c-26), 22.6 (c-27), 29.9 (c-28), 23.9 (c-29), 27.3 (c-30), 107.9 (c-31)

K4a: mp: 284-287° C.

[α]_(D) ²⁴+44° (c 0.5, Pyridine)

EI-MS m/z: 498[M]⁺, ¹³C-NMR (δ c): 35.9 (c-1), 37.1 (c-2), 217.3 (c-3), 53.5 (c-4), 43.9 (c-5), 24.5 (c-6), 121.5 (c-7), 143.7 (c-8), 144.9 (c-9), 37.9 (c-10), 119.0 (c-11), 36.9 (c-12), 45.9 (c-13), 50.0 (c-14), 45.0 (c-15), 77.3 (c-16), 58.2 (c-17), 18.5 (c-18), 23.3 (c-19), 49.4 (c-20), 179.9 (c-21), 32.2 (c-22), 34.1 (c-23), 157.0 (c-24), 34.9 (c-25), 22.8 (c-26), 22.7 (c-27), 19.4 (c-28), 67.5 (c-29), 26.9 (c-30), 107.8 (c-31)

K4b: mp: 230-232° C.

[α]_(D) ²⁴+38° (c 0.5, Pyridine)

EI-MS m/z: 542[M]⁺, ¹³C-NMR (δ c):

36.6 (c-1), 25.0 (c-2), 82.1 (c-3), 39.4 (c-4), 56.7 (c-5), 68.9 (c-6), 129.2 (c-7), 142.1 (c-8), 145.8 (c-9), 39.2 (c-10), 117.9 (c-11), 36.9 (c-12), 45.8 (c-13), 49.9 (c-14), 44.9 (c-15), 77.3 (c-16), 58.1 (c-17), 18.4 (c-18), 24.8 (c-19), 49.3 (c-20), 179.9 (c-21), 32.1 (c-22), 33.9 (c-23). 156.7 (c-24), 34.9 (c-25), 22.8 (c-26), 22.7 (c-27), 31.9 (c-28), 18.1 (c-29), 27.1 (c-30), 107.9 (c-31), 22.0 (CH₃COO—), 172.0 (CH₃ COO—)

K5: mp: 274-275° C.

[α]_(D) ²⁴+10° (c 0.5, Pyridine)

EI-MS m/z: 454[M]⁺, ¹³C-NMR (δ c):

36.8 (c-1) 29.6 (c-2), 79.0 (c-3), 38.6 (c-4), 50.6 (c-5), 24.3 (c-6), 122.1 (c-7), 143.6 (c-8), 147.3 (c-9), 37.2 (c-10), 117.5 (c-11), 34.1 (c-12), 45.0 (c-13), 51.3 (c-14), 30.8 (c-15), 28.1 (c-16), 48.9 (c-17), 17.4 (c-18), 23.7 (c-19), 49.9 (c-20), 179.9 (c-21), 32.4 (c-22), 27.5 (c-23), 124.3 (c-24), 132.7 (c-25), 26.6 (c-26), 18.6 (c-27), 29.2 (c-28), 17.1 (c-29), 26.7 (c-30)

K6a: mp: 248-250° C.

[a]_(D) ²⁴+63° (c 0.4, Pyridine)

EI-MS m/z: 498[M]⁺, ¹³C-NMR (δ c): 37.1 (c-1), 35.0 (c-2), 219.0 (c-3), 48.4 (c-4), 57.0 (c-5), 68.0 (c-6), 128.6 (c-7), 141.8 (c-8), 144.2 (c-9), 38.3 (c-10), 120.2 (c-11), 36.6 (c-12), 45.8 (c-13), 49.7 (c-14), 44.8 (c-15), 77.2 (c-16), 58.1 (c-17), 18.4 (c-18), 22.7 (c-19), 4.9.4 (c-20), 179.6 (c-21), 32.1 (c-22), 33.9 (c-23), 156.8 (c-24), 34.9 (c-25), 22.8 (c-26), 22.7 (c-27), 31.5 (c-28), 24.5 (c-269), 26.6 (c-30), 107.9 (c-31)

K6b: mp: 267-270° C.

[a]_(D) ²⁴+68° (c 0.3, Pyridine)

EI-MS m/z: 516[M]⁺, ¹³C-NMR (δ c): 34.4 (c-1), 29.4 (c-2), 74.1 (c-3), 42.6 (c-4), 87.7 (c-5), 133.1 (c-6), 134.7 (c-7), 79.6 (c-8), 145.8 (c-9), 42.1 (c-10), 120.7 (c-11), 36.8 (c-12), 49.1 (c-13), 48.7 (c-14), 42.7 (c-15), 76.8 (c-16), 57.6 (c-17), 19.0 (c-18), 29.3 (c-19), 49.1 (c-20), 179.6 (c-21), 32.2 (c-22), 33.9 (c-23), 156.8 (c-24), 34.9 (c-25), 22.9 (c-26), 22.7 (c-27), 25.1 (c-28), 20.3 (c-29), 20.8 (c-30), 107.9 (c-31)

The structures of K1 to K6 compounds are listed as follows:

Example 2

A powder was made of two kilograms of the China-grown Poria cocos (Schw) Wolf. By using the method disclosed in the China Patent Publication No. 1008183, a crude extract in an amount of 6.0 g was obtained, as shown in FIG. 2. Following the procedures described in Example 1, the crude extract was subjected to a silica gel column chromatography and eluted with a mixed solvent of dichloromethane:methanol=96:4 to obtain a PCM-E portion (2.0 g), and eluted with dichloromethane:methanol=90:10 and pure methanol in sequence to obtain a PCW-E portion (2.3 g).

The PCM-E and the PCW-E prepared in Example 2 were orally administered to animals in a dose of 40 mg/kg per day for the purpose of testing the effect of these two portions on growth of spleen cells (immunity cells) of the animals. The immune system of the animals will be enhanced, if the growth of spleen cells thereof are stimulated; and will be adversely affected, if the growth of spleen cells are inhibited, i.e. the spleen cells are killed due to toxicity.

The spleen cells were cultivated in vitro for five days before they were compared for cellular growth by means of MTT assay, which was determined by an immunological research method described hereinafter. The results are listed in the Table 1. The cellular growth of the spleen of the mice was apparently promoted on the third day and the fourth day in the wake of oral administration of PCM-E. Based on the statistic view point, there is no difference in terms of cellular growth among the control group and the experimental group. However, the number of the alive spleen cells of the mice was apparently lower than that of the control group on the third day and the fourth day in the wake of oral administration of PCW-E. In another words, the cellular growth of the spleen of the experimental group is apparently weakened as compared to the control group. This means that PCW-E is cytotoxic.

The implication is that the lanostane-containing low polarity portion, PCM, has no inhibitive effect on cellular growth of the spleen. In addition, the PCM portion has a promotive effect on cellular growth of the spleen. The secolanostane-containing high polarity portion (Rf<0.1) has an inhibitive effect on cellular growth of the spleen. The Poria extract of the present invention is devoid of the secolanostane fraction (PCW-E). On the contrary, the Poria extract obtained by the prior art methods contains the PCW-E capable of inhibiting cellular growth of the spleen.

TABLE 1 Effects of PCM-E and PCW-E on ceilular growth of spleen dose In vitro growth (day) component (mg/kg/day) 3rd day 4th day 5th day Control group 0.608 ± 0.042^(a ) 0.777 ± 0.141 0.515 ± 0.055 PCM-E 40 0.890 ± 0.195  0.857 ± 0.137 0.449 ± 0.083 PCW-E 40 0.245 ± 0.072*  0.451 ± 0.020** 0.344 ± 0.132 ^(a)data are mean ± S.E.M. of five experiments *is indicative of P < 0.05; **is indicative of P < 0.01

On the basis of the above two examples and the comparative results, the following facts are established.

The potent component of the Poria cocos (Schw) Wolf is a lanostane-containing low polarity portion, which is capable of enhancing immunity of human body.

A silica gel thin layer chromatography and a silica gel column chromatography can be employed to separate the promotive components from the inhibitive components of Poria cocos (Schw) Wolf.

The Poria extract obtained by the method of the present invention is devoid of the inhibitive components, which are secolanostanes and high polar molecules contained in the high polarity portion, PCW-E. For this reason, the method of the present invention is superior to the prior art methods.

The animal experiments of the present invention are carried out by an oral administration of PCM-E and PCW-E to animals. In another words, the administrations of PCM-E and PCW-E of the present invention take place in vivo other than in vitro. Because in vitro study of pharmaceutical compound on spleen cells dose not reflect actual effects of the pharmaceutical compound on cellular growth of the animal spleen in light of absence of interactions between cellular metabolism and the pharmaceutical compound. It is therefore readily apparent that the present invention is reliable and meaningful.

The Poria extract and the purified compounds from the Poria extract by chromatography prepared according to the methods of the present invention were tested by various immunity response experiments listed in the following. The PCM fraction of Poria extract and the lanostane compounds K1, K2, K3 and K4 purified therefrom in Example 1 exhibit immunity enhancement activities in immune cells (T cells/B cells) in the following immunity response tests, and, as shown in Table 2 to Table 10, K1, K2, K3 and K4 compounds are potent at dosage low than 2.5 or 5.0 mg/kg. 

1. A method of enhancing immunity of an individual comprising administering to the individual a therapeutically effective amount of a lanostane having the following chemical formula (I)

wherein R₁ is either H or CH₃; R₂ is OCOCH₃, ═O or OH; R₃ is H or OH; R₄ is —C(═CH₂)—C(CH₃)₂R_(a) wherein R_(a) is H or OH, or —CH═C(CH₃)—R_(b), wherein R_(b) is CH₃ or CH₂OH; R₅ is H or OH; and R₆ is CH₃ or CH₂OH.
 2. The method according to claim 1, wherein the lanostane (I) is


3. A method of enhancing immunity of an individual comprising administering to the individual a therapeutically effective amount of a Poria extract comprising 5-60% of a lanostane having the following chemical formula (I) by weight of the extract, and being substantially devoid of secolanostane:

wherein R₁ is either H or CH₃; R₂ is OCOCH₃, ═O or OH; R₃ is H or OH; R₄ is —C(═CH₂)—C(CH₃)₂R_(a), wherein R_(a) is H or OH, or —CH═C(CH₃)—R_(b), wherein R_(b) is CH₃ or CH₂OH; R₅ is H or OH; and R₆ is CH₃ or CH₂OH.
 4. The method according to claim 3, wherein the Poria extract is prepared by a method comprising the following steps: a) extracting metabolites, fermentation products or sclerotium of Poria cocos (Schw) Wolf by water, methanol, ethanol, or a mixed solvent thereof; b) concentrating the resulting liquid extract from step a); c) introducing the resulting concentrated substance from step b) into a silica gel column; d) eluting the silica gel column with an eluent having a low polarity, and collecting the resulting eluate; e) concentrating the eluate to form a concentrated eluate.
 5. The method of claim 4, wherein the concentrated eluate from step e) has a chromatographic value, Rf, not less than 0.1 in accordance with a thin layer chromatography, which is developed by a mixed solvent of dichloromethane:methanol=96:4 and is detected by an ultraviolet lamp and iodine vapor.
 6. The method of claim 4, wherein the extraction in step a) is carried out by using 95% ethanol.
 7. The method of claim 4, wherein the concentrated substance resulted from step b) is further extracted with a two-phase solvent containing methanol and n-hexane in a volumetric ratio of 1:1, a methanol layer is separated from the two-phase solvent extraction mixture, and the methanol layer is concentrated to form a concentrate, which is used as a feed to the silica gel column in step c).
 8. The method of claim 4, wherein the low polarity eluent is a mixed solvent containing dichloromethane and methanol in a volumetric ratio of 96.5:3.5.
 9. The method of claim 3, wherein the Poria extract comprises 10-20% of the lanostane (I).
 10. The method of claim 3, wherein the lanostane (I) is 